On 13/11/2007, Ed Siefker <ebs15242 from creighton.edu> wrote:
>> I got some really strange readings on my spec. For the inverse
> PCR I'm planning to do I ligated 4ug of genomic DNA in a volume
> of 4 ml with NEB's T4 ligase and buffer. After ligation I added
> 400ul of NaAc and 9ml EtOH. I precipitated it, washed with 70%
> EtOH, air dried, and resuspended in 10mM Tris.
just a quickie: did you use 4microgms of DNA in 4 millilitres of buffer?
.... I usually do my ligations in a total of 20 microL volume, so just
wondering.
On checking the concentration with a 1:25 dilution the readings
> were off the scale. I tried again with a 1:100 dilution the A260
> was still extremely high it was at least readable. The concentration
> it gave me was 13685ng/ul and the ratios 260/230 and 260/280 were
> both over 3. Adding more buffer pushed those ratios up to 6.
> Something is clearly very wrong here. I checked some .5ug/ul lambda
> hindiii ladder and it read ok. What could have possibly gotten in
> my DNA to do this?
so the concentration is actually ~13 mg/mL which is'nt so bad, is it?
od 260/280:
Double stranded nucleid acid typically has a ratio of 2.... 1.8 is very
good.
A single stranded nucleic acid/ oligo/RNA has a 260/280 ratio value ranging
from 1.0 to 2.5, depending on the base content. A higher percentage of C
and T bases have a lower value and those with A and G bases have a higher
value.
Methinks you want to check you calculations again:
Also, as a precaution, allow the DNA pellet to suspend in the buffer
completely....
Allow the UV lamp of the spec to warm up for at least 20 min.
Blank with the buffer you are diluting the sample with.
To verify your spec's fine, check another known sample.
These are a few things I can think of .....
Hope that helps,
Pow
_______________________________________________
> Methods mailing list
>Methods from net.bio.net>http://www.bio.net/biomail/listinfo/methods>